FBL Promotes LPS-Induced Neuroinflammation by Activating the NF-κB Signaling Pathway

Abstract

Purpose

Neuroinflammation occurs in response to central nervous system (CNS) injury, infection, stimulation by toxins, or autoimmunity. We previously analyzed the downstream molecular changes in HT22 cells (mouse hippocampal neurons) upon lipopolysaccharide (LPS) stimulation. We detected elevated expression of Fibrillarin (FBL), a nucleolar methyltransferase, but the associated proinflammatory mechanism was not systematically elucidated. The aim of this study was to investigate the underlying mechanisms by which FBL affects neuroinflammation.

Methods

RT-real-time PCR, Western blotting and immunofluorescence were used to assess the mRNA and protein expression of FBL in HT22 cells stimulated with LPS, as well as the cellular localization and fluorescence intensity of FBL. BAY-293 (a son of sevenless homolog 1 (SOS1) inhibitor), SR11302 (an activator protein-1 (AP-1) inhibitor) and KRA-533 (a KRAS agonist) were used to determine the molecular mechanisms underlying the effect of FBL. AP-1 was predicted to be the target protein of FBL by molecular docking analysis, and validation was performed with T-5224 (an AP-1 inhibitor). In addition, the downstream signaling pathways of FBL were identified by transcriptome sequencing and verified by RT-real-time PCR.

Results

LPS induced FBL mRNA and protein expression in HT22 cells. In-depth mechanistic studies revealed that when we inhibited c-Fos, AP-1, and SOS1, FBL expression decreased, whereas FBL expression increased when KRAS agonists were used. In addition, the transcript levels of inflammatory genes in the NF-kB signaling pathway (including CD14, MYD88, TNF, TRADD, and NFKB1) were elevated after the overexpression of FBL.

Conclusion

LPS induced the expression of FBL in HT22 cells through the RAS/MAPK signaling pathway, and FBL further activated the NF-kB signaling pathway, which promoted the expression of relevant inflammatory genes and the release of cytokines. The present study reveals the mechanism by which FBL promotes neuroinflammation and offers a potential target for the treatment of neuroinflammation.

Keywords:

• FBL
• neuroinflammation
• LPS
• molecular docking
• transcriptome sequencing

Abbreviations

LPS, lipopolysaccharide; CNS, central nervous system; NF-kB, nuclear factor kB; AP-1, activating protein-1; NFAT, nuclear factor of activated T cell; STAT, signal transducer and activator of transcription; MAPKs, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; NLRP3, NLR family pyrin domain containing 3; FBL, Fibrillarin; snoRNP, small nucleolar ribonucleoprotein; snoRNAs, small nucleolar ribonucleic acids; NFTs, neurofibrillary tangles; NMDA, N-methyl-D-aspartic acid; Aβ, amyloid plaques; SOS1, son of sevenless homolog 1; RTKs, receptor tyrosine kinases; Grb2, growth factor receptor-bound protein 2; LBP, lipopolysaccharide-binding protein; MYD88, myeloid differentiation factor 88; CD14, cluster of differentiation 14; IL, interleukin; TNF-α, tumor necrosis factor α; CSF, cerebrospinal fluid; real-time RT-PCR, real-time reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Data Sharing Statement

The data used in this study are available from the corresponding author upon reasonable request.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (Nos. 82171321, 82171363, and 82371381).